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Bacterial plasmids are supercoiled loops of DNA that are typically in the range of thousands of base pairs in length—relatively small compared to the linear bacterial chromosomal DNA (chDNA), which is typically millions of base pairs in length. If the double-stranded DNA of the plasmid DNA (pDNA) loop is nicked, the loop will untwist to form open-circular (OC) loops of DNA. These differences in topology and length give substantially different electrophoretic behaviours. Once purified, pDNA can be used in a wide variety of downstream applications such as cloning, sequencing, PCR, expression of proteins, transfection, and gene therapy. In addition, the rapid and inexpensive development of new plasmid purification methods may also provide an efficient means to test the efficiency of transfections and confirm the existence or absence of given mutations in transfected cell experiments. The preparation of plasmids is therefore a key technology for many applications in the fields of molecular biology, genetics, biochemistry and cell biology.
Among the multitude of plasmid purification methods, the alkaline lysis method is widely used to prepare bacterial plasmids in highly purified form. This method relies on lysing of bacterial cells under alkaline conditions and precipitation of the chDNA. Removal of chDNA through centrifugation allows subsequent purification of the pDNA. Bacterial plasmid mini-preps have been made easier through the availability of commercial kits that provide prepared solutions, although the procedure still takes about an hour. While the speed is faster, these kits considerably increase the per-preparation cost involved. Most importantly, these kits require large amounts of sample and their integration into a lab-on-chip format is problematic in terms of speed, the need for complex fluid manipulations, filtering and centrifuging. While mini-prep methods are commonplace for bacteria and yeast, they are not commonly used for higher eukaryotes, mostly because of the large amounts of material and the expense that would be needed for such an approach.
pDNA molecules can exist either in a supercoiled (SC) conformation, a loop that contains sufficient twists that the DNA bunches into a ball, or in an open circular (OC) conformation, whereby nicking of the SC loop relaxes the DNA into an untwisted loop. One of the most common means of analyzing plasmids electrophoretically is in agarose, whose structure is known to consist of a random, three-dimensional network of long, straight, connected fibers, each made of 10 to 30 double helices (Arnott, S. et al. J. Mol. Biol 90:269 (1974); Waki, S. et al Biopolymer 21:1909 (1982)). At electric fields above a critical value, trapping of both the SC and OC forms of plasmid DNA has been observed. Two models are commonly used to explain this trapping—the impalement model and the lobster trap model (Akerman, B. et al. Electrophoresis 23:2549 (2002)). The impalement model proposes that circular DNA is trapped by becoming caught or impaled upon the free end of a fiber in such a network. In contrast, the lobster trap model posits that the DNA is caught in constricted ‘dead-ends’ in the sieving matrix.
Akerman (Akerman, B. Biophys J 74:3140 (1998)) used an elegant method based on linear dichroism (LD) to study the behaviour of SC and OC DNA in polyacrylamide (PA) and agarose gels under fields of 7.5 to 22.5 V/cm in TBE buffer. Although the supercoiled (SC DNA or scDNA) was not trapped in the agarose gel, in PA the SC DNA showed a “rapidly fading smear” that extended 3-4 mm from the loading well, whereas the OC DNA was trapped immediately. The SC smear was attributed to less efficient trapping of the SC DNA as compared to the OC DNA. Akerman proposed that the circular DNA was trapped by impalement upon protruding fibres within the PA gel, with the OC DNA forming an extended, open loop and the SC DNA forming an extended, twisted loop (Akerman, B. Biophys J 74:3140 (1998)). Akerman estimated a characteristic dangling fibre length of between 8.7 and 33 nm for PA.
LD measurements of the time constants of DNA orientation under an electric field, was used this as an indication of the degree of trapping, finding the rate of DNA being trapped was proportional to E−n, where n was approximately 2. Transport effects were expected to give a dependence of E−1 and the additional contribution of E−1 was attributed to having more trapping sites available at higher fields (shorter gel fibres will be able to trap at higher fields). The exponent of this additional contribution was expected to vary depending on the distribution in lengths of dangling fibres. To explain the lower trapping rate of the SC DNA, Akerman suggested that the supercoiling of the DNA reduces the size of the holes in the DNA coil, lowering the probability of penetration by a gel fibre. In addition, the SC DNA presents a smaller cross-sectional area available for impalement. Although that work was limited in the range of SC DNA studied (two sizes, 2926 and 5386 bp), Akerman found that the larger SC DNA was immobilized faster than the smaller. To explain the absence of trapping within agarose, Akerman suggested (and supported with literature values) that the holes in the supercoiled structure were too small to be penetrated by the agarose gel fibres (diameter of 3-9 nm) but not by the fibres of the PA (−0.1 nm in diameter).
Although the relationship between the size of trapped linear DNA and the electric field (E) is well known, and underlies such techniques as pulsed field gel electrophoreses, this trapping relationship for circular DNA is not nearly as well understood. As described above, considerable progress has been made in understanding the behaviour of OC and SC DNA, but in some cases this work has been complicated by the use of imaging techniques that inadvertently nick the fragile SC DNA, thereby converting it to OC DNA